Activation of αIIbβ3 is a sufficient but also an imperative prerequisite for activation of α2β1 on platelets

Abstract

Platelet integrins ␣ 2 ␤ 1 and ␣ IIb ␤ 3 play critical roles in platelet adhesion and thrombus formation after vascular injury. On resting platelets, both integrins are in a low-affinity state. However, agonist stimulation results in conformational changes that enable ligand binding that can be detected with conformation dependent monoclonal antibodies (mAbs). By using such conformation-dependent mAbs, we could demonstrate that activation of integrin ␣ IIb ␤ 3 is not only sufficient, but also a prerequisite for ␣ 2 ␤ 1 activation. Compared with platelets in plasma, stimulation of washed platelets resulted in only a minor activation of ␣ 2 ␤ 1 , as detected with the activation-sensitive mAb IAC-1. Addition of fibrinogen to stimulated washed platelets greatly potentiated activation of this integrin. Also, treatment of ␣ IIb ␤ 3 with the ligand-mimetic peptide RGDS, resulting in outside-in signaling, led to a powerful ␣ 2 ␤ 1 activation, even in the absence of overall platelet activation, involving tyrosine kinase activity but no protein kinase C activation. The absolute necessity of ␣ IIb ␤ 3 for proper ␣ 2 ␤ 1 activation on platelets was demonstrated by using the ␣ IIb ␤ 3 antagonist aggrastat, which was able to completely abolish ␣ 2 ␤ 1 activation, both under static and flow conditions. In addition, analogous experiments with Glanzmann platelets lacking ␣ IIb ␤ 3 confirmed the indispensability of ␣ IIb IntroductionIntegrins are a large family of heterodimeric transmembrane receptors, each consisting of an ␣ and ␤ subunit, and are key effectors of cell growth, migration, differentiation, and survival. 1,2 Integrins possess the unique ability to signal across the plasma membrane in both directions, and since most integrins are not constitutively active, they are expressed on the cell surface as low-affinity receptors. When cells become activated, cytosolic proteins can bind to the cytoplasmic domains of integrins and as a consequence, the integrins are turned into their high-affinity state ("inside-out" signaling). In a process called "outside-in" signaling, ligand-binding of integrins then again activates intracellular pathways via their cytoplasmic domains, which are connected to the cytoskeleton and are associated with several intracellular signaling molecules. Important signaling modulators necessary for the generation of outside-in signals are members of the Src family protein tyrosine kinases, with c-Src as the initiating molecule due to its constitutive interaction with, for example, ␤ 3 integrins. 3 Such outside-in signals also result in conformational alterations of the integrin (also designated integrin activation), and subsequently these activated integrins can trigger another process of inside-out signaling. 4 This integrin activation upon conformational changes is often defined as an increase in integrin "affinity" for its ligand, and has been the topic of many studies. [4][5][6] In addition, cell activation also promotes clustering of integrins contributing to the "avidity" or "valency" regulation ...

Distinct spatio-temporal Ca2+ signaling elicited by integrin α2β1 and glycoprotein VI under flow

“…4,5 Our evidence supports the existence of a functional synergy between ␣2␤1 and GPVI, but not simply to the effect that GPVI provides primary signals subsequently amplified by ␣2␤1. 31,37 Not only did we find that ␣2␤1 can signal independently of GPVI or ␣IIb␤3 (GPIIb-IIIa), 38 as clearly confirmed by the induction of ␣-like Ca 2ϩ peaks on selective ␣2␤1 ligation by the GFOGER peptide, but also that GPVI functions are facilitated by preceding ␣-like Ca 2ϩ elevations dependent on ␣2␤1 engagement. Such conclusions do not exclude the possibility that ␣2␤1 ligand-binding affinity is further enhanced after GPVI interaction with collagen and/or ␣IIb␤3 activation.…”

Cell Adhesion Mechanisms in Platelets

“…84 A recent study that used an antibody that recognizes activation-induced conformational changes in ␣2␤1 suggested that the activation of this integrin is dependent on the previous activation of ␣IIb␤3. 85 However, the underlying mechanisms have not been identified and it remains to be determined whether the detected conformational changes indeed reflect "activation" of the integrin.…”

Platelet receptors and signaling in the dynamics of thrombus formation

“…33,34 Like other integrins, conformational activation of α2β1 increases its affinity for collagen, and seems to require inside-out signaling events that might be driven by engagement of GP VI with collagen 23 and/or by activation of αIIbβ3. 35 Recent findings indicate that the mechanism of activation of α2β1 is similar to that of other integrins and involve unclasping of the corresponding transmembane domain upon interaction of the α1 cytoplasmic tail with talin and kindlin-3. 36 Moreover, collagen binding to α2β1 also triggers outsidein signaling resembling that induced by GP VI, 23 that reinforces platelet activation.…”